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Interstitial cystitis antiproliferative factor (APF) as a cell-cycle modulator.

Rashid HH, Reeder JE, O'Connell MJ, Zhang CO, Messing EM, Keay SK - BMC Urol (2004)

Bottom Line: Cell cycle phase fractions were calculated from the DNA frequency distributions and compared by two-way analysis of variance (ANOVA).APF exposure produced statistically significant increases in the proportion of tetraploid and hypertetraploid cells compared to mock control preparations, suggesting a G2 and/or M phase cell cycle block and the production of polyploidy.APF has a specific effect on cell cycle distributions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, University of Rochester, Rochester, New York, USA. hani_rashid@urmc.rochester.edu

ABSTRACT

Background: Interstitial cystitis (IC) is a chronic bladder disorder of unknown etiology. Antiproliferative factor (APF), a peptide found in the urine of IC patients, has previously been shown to decrease incorporation of thymidine by normal bladder epithelial cells. This study was performed to determine the effect of APF on the cell cycle of bladder epithelial cells so as to better understand its antiproliferative activity.

Methods: Explant cultures from normal bladder biopsy specimens were exposed to APF or mock control. DNA cytometry was performed using an automated image analysis system. Cell cycle phase fractions were calculated from the DNA frequency distributions and compared by two-way analysis of variance (ANOVA).

Results: APF exposure produced statistically significant increases in the proportion of tetraploid and hypertetraploid cells compared to mock control preparations, suggesting a G2 and/or M phase cell cycle block and the production of polyploidy.

Conclusions: APF has a specific effect on cell cycle distributions. The presence of a peptide with this activity may contribute to the pathogenesis of interstitial cystitis through disruption of normal urothelial proliferation and repair processes.

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Representative DNA frequency distributions DNA frequency distributions from 10 μl mock treated (top panel) and 10 μl APF treated (bottom panel) normal bladder epithelial cell cultures show an APF associated increase in the proportion of cells with tetraploid DNA value. The presence of cells with octaploid DNA values suggests the cultures contain a mixture of cycling diploid and tetraploid cells.
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Figure 1: Representative DNA frequency distributions DNA frequency distributions from 10 μl mock treated (top panel) and 10 μl APF treated (bottom panel) normal bladder epithelial cell cultures show an APF associated increase in the proportion of cells with tetraploid DNA value. The presence of cells with octaploid DNA values suggests the cultures contain a mixture of cycling diploid and tetraploid cells.

Mentions: All specimens were cytocentrifuged onto slides using a CytoTek Cytocentrifuge at 1,000 RPM for 5 minutes, fixed in 10% Böhm-Springer fixative, hydrolyzed for 45 minutes in 5 N hydrochloric acid and the DNA stained by Feulgen reaction using thionin according to Perceptronix Medical Incorporated (PMI) standard protocol. The Fuelgen stained nuclei were analyzed on a PMI Cytosavant image analysis instrument, which was programmed to scan each slide and acquire 2,000 single nuclei. Debris and cell clumps were rejected using optical density and morphometric features. DNA content was calculated from sum optical density. Diploid control cells were used for validation and identification of G1 cell populations for normalization. Morphometric features for selecting single, whole nuclei included area, sphericity, and eccentricity. After acquisition, cell image galleries were reviewed to ensure that only data from whole, single nuclei were retained in the frequency distribution file. The frequency and cumulative frequency of nuclei were plotted for the calculated sum optical density (DNA content) (Figures 1 and 2). The boundaries of the cell cycle phases were identified by the inflection points in the cumulative frequency distribution and cell subpopulation fractions were calculated.


Interstitial cystitis antiproliferative factor (APF) as a cell-cycle modulator.

Rashid HH, Reeder JE, O'Connell MJ, Zhang CO, Messing EM, Keay SK - BMC Urol (2004)

Representative DNA frequency distributions DNA frequency distributions from 10 μl mock treated (top panel) and 10 μl APF treated (bottom panel) normal bladder epithelial cell cultures show an APF associated increase in the proportion of cells with tetraploid DNA value. The presence of cells with octaploid DNA values suggests the cultures contain a mixture of cycling diploid and tetraploid cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC411044&req=5

Figure 1: Representative DNA frequency distributions DNA frequency distributions from 10 μl mock treated (top panel) and 10 μl APF treated (bottom panel) normal bladder epithelial cell cultures show an APF associated increase in the proportion of cells with tetraploid DNA value. The presence of cells with octaploid DNA values suggests the cultures contain a mixture of cycling diploid and tetraploid cells.
Mentions: All specimens were cytocentrifuged onto slides using a CytoTek Cytocentrifuge at 1,000 RPM for 5 minutes, fixed in 10% Böhm-Springer fixative, hydrolyzed for 45 minutes in 5 N hydrochloric acid and the DNA stained by Feulgen reaction using thionin according to Perceptronix Medical Incorporated (PMI) standard protocol. The Fuelgen stained nuclei were analyzed on a PMI Cytosavant image analysis instrument, which was programmed to scan each slide and acquire 2,000 single nuclei. Debris and cell clumps were rejected using optical density and morphometric features. DNA content was calculated from sum optical density. Diploid control cells were used for validation and identification of G1 cell populations for normalization. Morphometric features for selecting single, whole nuclei included area, sphericity, and eccentricity. After acquisition, cell image galleries were reviewed to ensure that only data from whole, single nuclei were retained in the frequency distribution file. The frequency and cumulative frequency of nuclei were plotted for the calculated sum optical density (DNA content) (Figures 1 and 2). The boundaries of the cell cycle phases were identified by the inflection points in the cumulative frequency distribution and cell subpopulation fractions were calculated.

Bottom Line: Cell cycle phase fractions were calculated from the DNA frequency distributions and compared by two-way analysis of variance (ANOVA).APF exposure produced statistically significant increases in the proportion of tetraploid and hypertetraploid cells compared to mock control preparations, suggesting a G2 and/or M phase cell cycle block and the production of polyploidy.APF has a specific effect on cell cycle distributions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, University of Rochester, Rochester, New York, USA. hani_rashid@urmc.rochester.edu

ABSTRACT

Background: Interstitial cystitis (IC) is a chronic bladder disorder of unknown etiology. Antiproliferative factor (APF), a peptide found in the urine of IC patients, has previously been shown to decrease incorporation of thymidine by normal bladder epithelial cells. This study was performed to determine the effect of APF on the cell cycle of bladder epithelial cells so as to better understand its antiproliferative activity.

Methods: Explant cultures from normal bladder biopsy specimens were exposed to APF or mock control. DNA cytometry was performed using an automated image analysis system. Cell cycle phase fractions were calculated from the DNA frequency distributions and compared by two-way analysis of variance (ANOVA).

Results: APF exposure produced statistically significant increases in the proportion of tetraploid and hypertetraploid cells compared to mock control preparations, suggesting a G2 and/or M phase cell cycle block and the production of polyploidy.

Conclusions: APF has a specific effect on cell cycle distributions. The presence of a peptide with this activity may contribute to the pathogenesis of interstitial cystitis through disruption of normal urothelial proliferation and repair processes.

Show MeSH
Related in: MedlinePlus